High-sensitive fluorescent energy transfer assay using fluorescent amino acids and fluoresent proteins

Abstract

The disclosure provides method and composition utilizing fluorescent amino acids and fluorescent proteins comprising a moiety capable of undergoing FRET. The methods and compositions of the disclosure are useful in analyzing protein structure and function, and screening molecular inhibitors.

1 . A polypeptide comprising:
 at least one first fluorescent amino acid comprising NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl); and   at least one second fluorescent amino acid comprising a coumarin fluorescent amino acid or derivative thereof.   
     
     
         2 - 6 . (canceled) 
     
     
         7 . The polypeptide of  claim 1 , wherein the polypeptide is synthesized. 
     
     
         8 . The polypeptide of  claim 1 , wherein the polypeptide is genetically expressed. 
     
     
         9 . The polypeptide of  claim 1 , wherein each of the at least one first and second fluorescent amino acids are separated by 1, 2, 3, 4, 5, 6, 7, 8 or more amino acids and no more than about 10 nm. 
     
     
         10 . The polypeptide of  claim 1 , wherein the at least one first and second fluorescent amino acids are separated by 4 to 6 amino acids. 
     
     
         11 . (canceled) 
     
     
         12 . The polypeptide of  claim 1 , wherein the at least one first and second fluorescent amino acids are separated by a protease cleavage site. 
     
     
         13 - 19 . (canceled) 
     
     
         20 . A method of determining a structure of a polypeptide comprising:
 providing a polynucleotide encoding the polypeptide, wherein the polynucleotide comprises at least two codons that results in the incorporation of at least two chromophore or fluorescent amino acid upon translation;   translating the polynucleotide to obtain a labeled polypeptide comprising the at least two chromophore or fluorescent amino acid,   wherein the at least two chromophore or fluorescent amino acids comprise different moieties comprising an acceptor or donor chromophore or fluorophore moieties that undergo Förster resonance energy transfer (FRET), and   identifying FRET in the polypeptide, wherein the presence of FRET is indicative that the at least two amino acids are within a selected distance from one another, thereby providing a structure of the polypeptide.   
     
     
         21 . The method of  claim 20 , carried out in a cell-free system. 
     
     
         22 . The method of  claim 20 , carried out in a cell. 
     
     
         23 . The method of  claim 20 , wherein the acceptor is a quenching moiety. 
     
     
         24 . A FRET measurement system comprising:
 at least a first amino acid comprising a fluorophore comprising NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl); and   at least a second amino acid comprising a fluorophore comprising a coumarin fluorescent amino acid wherein the first and second amino acids are incorporated into a polypeptide.